JP6401712B2 - Bovine colostrum enzyme-treated product, method for producing the same, composition, and food and drink - Google Patents

Description

  The present invention relates to a pharmaceutical composition comprising a bovine colostrum enzyme-treated product and a method for producing the same, which are useful for the treatment and prevention of diseases such as cancer and infectious diseases.

  Macrophages are responsible for processing waste products in the body and protecting against pathogens such as microorganisms and viruses and tumor cells. It also has a function as an effector of cellular immunity through presentation of antigen to T cells and production of interleukin-1. Therefore, it is important to activate macrophages for the treatment and prevention of cancer and infectious diseases and the like, and it is possible to treat and prevent cancer and infectious diseases by activating macrophages.

  Examples of factors that activate macrophages include interferon, and clinical applications thereof are also being attempted. In addition, it is known that certain polysaccharides have immunostimulatory activity, and some of these are expected to be developed as antiviral agents and anticancer agents (Patent Document 1 or 2).

  Patent Document 3 describes that an enzyme-treated product of human serum (β-galactosidase, or β-galactosidase and sialidase) has a macrophage activating action.

Japanese Patent Laid-Open No. 05-097695 Japanese Patent Publication No. 06-099314 International Publication No. 2013/038997

  The present invention provides a bovine colostrum enzyme-treated product, a production method thereof, a composition comprising the same, and various products, which are useful for treatment, prevention, amelioration, maintenance of remission of diseases such as cancer and infectious diseases. It is what.

  As a result of intensive studies, the present inventors have shown that bovine colostrum exhibits an excellent macrophage activation effect when it is contacted with a specific enzyme, that is, β-galactosidase, or β-galactosidase and sialidase, and treated with the enzyme. The present invention was completed through repeated headings and further studies.

That is, the present invention
[1] A method for producing an enzyme-treated bovine colostrum comprising a step of contacting bovine colostrum with β-galactosidase,
[2] The production method of the above-mentioned [1], further comprising a step of contacting bovine colostrum with sialidase,
[3] A bovine colostrum enzyme-treated product obtained by the production method according to [1] or [2] above,
[4] For single administration, 0.02 μg to 40 mg, preferably 0.02 μg to 20 mg, more preferably 0.2 μg to 20 mg, more preferably 2 μg to 20 mg, more preferably 20 μg to 20 mg, more preferably 200 μg to 10 mg, more preferably 200 μg to 2 mg of the protein treated with bovine colostrum enzyme according to the above [3],
[5] A pharmaceutical composition comprising the bovine colostrum enzyme-treated product according to [3] or [4] above,
[6] The pharmaceutical composition according to the above [5], which is for anticancer or antiinfective use,
[7] A composition for food or drink comprising the bovine colostrum enzyme-treated product according to [3] or [4] above,
[8] A food or drink comprising the composition for food or drink according to [7] above,
About.

  Since the bovine colostrum enzyme-treated product of the present invention has an excellent macrophage activating action, particularly an intestinal macrophage activating action, it is useful for the treatment and prevention of diseases such as cancer and infectious diseases. It can be used as a disease agent.

  Moreover, if this bovine colostrum enzyme-treated product is used, it is possible to provide a quasi-drug useful for prevention, improvement, and maintenance of remission of the above-mentioned diseases, and a composition for food and drink and a food and drink.

  Furthermore, since the bovine colostrum enzyme-treated product according to the present invention can be prepared by treating bovine colostrum with β-galactosidase, or β-galactosidase and sialidase, it has an advantage of being simple and low-cost.

It is a drawing substitute photograph which shows the mode of the macrophage dye | stained with Giemsa. It is a graph which shows the result about the macrophage phagocytic activity using 0.5% opsonized SRBC of each test substance. It is a drawing substitute photograph which shows the result of electrophoresis.

<Bovine colostrum>
The bovine colostrum used in the present invention refers to milk secreted by calves by the 10th day after calf delivery, preferably by 7 days, more preferably by the 5th day. is there. In the present invention, any bovine colostrum can be used regardless of the type of cow such as Holstein and Japanese Black.

<Enzyme>
The β-galactosidase used in the present invention is not particularly limited, and any known type can be used. Examples thereof include those derived from Escherichia coli and those derived from bovine liver. Examples of commercially available products include catalog No. of Wako Pure Chemical Industries, Ltd. 072-04141, G1875 of SIGMA-ALDRICH, and the like.
In the present invention, β-galactosidase may be used alone or in combination of two or more.

The sialidase used in the present invention is not particularly limited, and any known type can be used. As such, for example, those derived from Clostridium perfringenes, those derived from Streptococcus 6646K, those derived from Vibrio cholerae, Arthrobacter ureafaciens (Arthrobacter ureafaciens) The thing of origin etc. are mention | raise | lifted. Examples of commercially available products include SIGMA-ALDRICH product numbers (Sigma Prod. Nos.) N2876, N2133, N2904, N3001, N5631, Biochemical Biobusiness Code Number 120052, BioLabs Catalog numbers (Catalog #) P0720L, P0720S, and the like.
In the present invention, the sialidase may be used alone or in combination of two or more.

<Enzyme treatment>
In the present invention, the contact (enzyme treatment) of bovine colostrum with β-galactosidase or sialidase makes contact with a sufficient amount of enzyme for a sufficient time, so that the enzyme reaction further proceeds substantially. It is preferable to carry out to such an extent that it does not. For this purpose, although it depends on the kind of enzyme, for example, catalog No. of Wako Pure Chemical Industries, Ltd. as β-galactosidase. When using 072-04141, it is sufficient to use 65 mU of enzyme for 100 μl of bovine colostrum. For example, when using the product number (N2876) of SIGMA-ALDRICH as sialidase, it is sufficient to use 65 mU of enzyme for 100 μl of bovine colostrum. In this case, it is sufficient to perform the enzyme treatment for 3 hours.

Enzymatic treatment can be carried out in any vessel by adding these enzymes to bovine colostrum, but is commonly used in this field to adjust the total protein concentration in bovine colostrum if desired. Buffer may be added. Examples of such a buffer include physiological saline, phosphate buffered saline (SPB), Ringer's solution, and the like.
The temperature of the enzyme treatment is not particularly limited as long as the enzyme exhibits activity, but it is usually around 37 ° C. where the enzyme exhibits high activity.

The enzyme treatment is terminated by inactivating the enzyme by heating (heat treatment). Such heat treatment is not particularly limited as long as the enzyme can be inactivated. For example, the heat treatment can be performed by heating at a temperature around 60 ° C. for about 10 minutes.
The specimen after the heat treatment may be concentrated if desired. The concentration can be performed using a commercially available device, for example, a centrifugal concentrator (for example, 10000MWCO YM-10 manufactured by MILLIPORE).

  The enzyme treatment can also be performed using an enzyme (immobilized enzyme) immobilized on a solid phase. A method for immobilizing an enzyme on a solid phase is known to those skilled in the art. For example, β-galactosidase and / or sialidase can be immobilized on agarose beads with a coupling agent such as cyanogen bromide. Examples of such immobilized enzymes include immobilized β-galactosidase G3M (Mo Bi Tec, # A3102), neuraminidase agarose Clostridium perfringens (manufactured by Welsh bacteria) (manufactured by SIGMA-ALDRICH, product number: N5254). ) Etc. are commercially available. The advantage of using an immobilized enzyme is that the enzyme can be recovered without being deactivated by heat treatment after the enzyme treatment, and foreign substances (proteins such as enzymes deactivated by heat treatment) by such recovery It is possible to reduce the existence of.

<Bovine colostrum enzyme-treated product>
The thus obtained bovine colostrum enzyme-treated product of the present invention may be further freeze-dried to form a solid or powder. Such a bovine colostrum enzyme-treated product is a novel composition useful for the treatment, prevention, improvement, maintenance of remission of diseases such as cancer and infectious diseases.

<Pharmaceutical compositions and pharmaceuticals>
The bovine colostrum enzyme-treated product of the present invention can be prepared as a pharmaceutical composition as it is or by appropriately blending a pharmaceutically acceptable adjuvant (carrier). As such pharmaceutically acceptable adjuvants, any of those commonly used in this field can be suitably used. Specific examples include diluents, stabilizers, preservatives, buffering agents. , Excipients, binders, preservatives, disintegrants, lubricants, flavoring agents and the like. These adjuvants are appropriately blended according to the dosage form of the pharmaceutical composition.

  The pharmaceutical composition of the present invention can be formulated into an appropriate dosage form into a pharmaceutical product. Such a dosage form is not particularly limited, and may be an oral preparation or a parenteral preparation. Examples of parenteral preparations include injections, infusions, nasal drops, ear drops, suppositories, enteral nutrients and the like. For example, examples of the injection include those in administration forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, intraperitoneal injection, etc. Among them, intramuscular injection is preferable. On the other hand, as oral preparations, powders, granules, tablets (including sublingual tablets), capsules, pills, intestinal solvents, liquids for internal use (including suspensions, emulsions, syrups, etc.), inhalants Etc.

  The dose of the bovine colostrum enzyme-treated product of the present invention varies depending on the age, sex, weight and symptoms, administration method, etc. of the subject of administration, but typical examples include, for example, this bovine colostrum enzyme-treated product. The total amount of protein to be added is about 0.02 μg or more, preferably about 0.2 μg or more, more preferably about 2 μg or more, more preferably about 20 μg or more, more preferably about 200 μg or more per kg of body weight per administration. And about 40 mg or less, preferably about 20 mg or less, more preferably about 13 mg or less, more preferably about 10 mg or less, more preferably 2 mg or less. Preferred dosage ranges include, for example, from about 0.02 μg to about 40 mg, preferably from about 0.02 μg to about 20 mg, more preferably from about 0.2 μg to about 20 mg, more preferably from about 2 μg to about 20 mg, more preferably Is about 20 μg to about 20 mg, more preferably about 200 μg to about 10 mg, more preferably about 200 μg to about 2 mg. Other preferred ranges are from about 1 mg to about 40 mg, preferably from about 2 mg to about 20 mg, more preferably from about 3 mg to about 13 mg. In the present specification, the amount of protein is calculated based on the protein concentration determined by the absorbance at a wavelength of 570 nm.

  When the pharmaceutical composition of the present invention is administered at a dose per administration as described above, a typical administration interval and administration frequency are 1 to 2 times / day. The dose and the administration interval can be appropriately changed within a range in which the total amount of protein to be administered is equivalent with the total amount of protein contained in the pharmaceutical composition as an index.

  The pharmaceutical composition of the present invention has a macrophage activation effect. Therefore, the pharmaceutical composition of the present invention can be used as a therapeutic or prophylactic agent for diseases that can be treated or prevented by these actions. Such diseases include cancer and infectious diseases.

  Cancer includes any of carcinoma, sarcoma, and other malignant tumors, such as skin cancer, bronchial cancer, lung cancer, non-small cell lung cancer, breast cancer, ovarian cancer. , Tongue cancer, pharyngeal cancer, esophageal cancer, stomach cancer, small intestine cancer, colon cancer, rectal cancer, colon cancer, liver cancer, pancreatic cancer, kidney cancer, renal cell cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, Wilms Tumor, malignant melanoma, meningioma, neuroblastoma, osteosarcoma, Kaposi sarcoma, lymphoma, leukemia and the like. In this specification, the term “cancer” includes these malignant tumors as well as their metastases.

  Examples of infectious diseases include viral infections, bacterial infections, and the like. Specifically, in addition to HIV infection, AIDS, hepatitis B, hepatitis C, herpes, influenza, pneumonia, tuberculosis, Examples include EB virus infection.

  The pharmaceutical composition of the present invention can be used in combination with other anticancer agents and antiinfective agents. When used in combination, the dosage of the pharmaceutical composition of the present invention is appropriately adjusted in consideration of the efficacy, effects and dosage of the other drugs.

<Quasi-drug>
The bovine colostrum enzyme-treated product of the present invention can be prepared not only as a pharmaceutical as described above but also as a quasi-drug. The quasi drug can contain the above-mentioned adjuvants and the like as necessary. Quasi-drugs can take various forms such as solutions, suspensions, syrups, granules, creams, basets, jellies, etc. You can also The amount of bovine colostrum enzyme-treated product to be used as a quasi-drug is not particularly limited, but can be appropriately set with reference to the dose in the case of the pharmaceutical product.

<Composition for food and drink, food and drink>
The bovine colostrum enzyme-treated product of the present invention is variously used in foods and drinks such as the above-mentioned adjuvants, sweeteners, spices, seasonings, preservatives, preservatives, bactericides, antioxidants, etc. It can be set as the composition for food-drinks by mix | blending an additive suitably, Furthermore, this food-drinks composition can be further processed, and it can be set as the food-drinks containing this. The composition for food or drink or food or drink can take various forms such as solution, suspension, syrup, granule, cream, basto, jelly, etc. It can also be formed into a shape. In addition, the food and drink can take various forms such as bread, noodles, confectionery, beverages, soups, and processed foods. Preparation of the composition for food / beverage products and food / beverage products can be carried out according to a conventional method.

  Since the bovine colostrum enzyme-treated product of the present invention has an effect on the diseases as described above, the composition for food and beverage and the food and beverage of the present invention can prevent, improve, and maintain remission of such diseases. Can be effective. In this case, the amount of the bovine colostrum enzyme-treated product in the composition for food or drink of the present invention or food or drink is not particularly limited, but with reference to the dosage in the case of the above pharmaceutical product, Can be set. Preferable specific examples of the use amount of the bovine colostrum enzyme-treated product include, for example, about 1 mg to about 40 mg, preferably about 2 mg to about 20 mg, more preferably about 3 mg to about 13 mg per kg of body weight per meal. Range.

  Such foods and drinks of the present invention can be so-called health foods, health drinks, foods for specified health use, functional foods, dietary supplements (supplements), and feeds for animals other than humans.

  Such pharmaceuticals, quasi-drugs, and foods and drinks according to the present invention absorb the bovine colostrum enzyme-treated product, which is an active ingredient, from the digestive tract, preferably from the oral cavity or the intestinal tract (for example, the above-mentioned tongue It is preferably in the form of a tablet or enteric solvent. This is because an effect of directly activating macrophages in the oral cavity or intestinal tract can be expected. In particular, it is known that an abundant number of macrophages, which are said to be the largest in the body, exist in the Peyer's patch in the gut-associated lymphoid tissue (GALT).

  The present invention will be described in detail based on examples, but the present invention is not limited to these examples.

1. Preparation of bovine colostrum enzyme-treated product (1)
1 mg of solid bovine colostrum (Now Foods “Colostrum Powder”) was dissolved in 1 ml of 1 × PBS, and β-galactosidase (manufactured by Wako Pure Chemical Industries, Ltd., Catalog No. 072-04141) was dissolved in 100 μl of the colostrum. , 10 mU / μl) 6.5 μl, sialidase (SIGMA-ALDRICH, N2876, 10 mU / μl) 6.5 μl, and 100 mM SPB (15.601 g NaH ₂ PO ₄ .2H ₂ O and 35.814 g Na ₂ HPO ₄ · 12H ₂ O was dissolved in 500 ml of distilled water to prepare 200 mM SPB (pH 7.0), which was diluted to 100 mM SPB.) 87 μl was added and incubated at 37 ° C. for 3 hours. did. After the incubation, 200 μl of 100 mM SPB was further added and heat-treated at 60 ° C. for 10 minutes. After heat treatment, the sample was concentrated with a microcon (10000MWCO YM-10, MILLIPORE), and the protein concentration was determined by measuring absorbance at a wavelength of 570 nm (using a calibration curve prepared for BSA (bovine serum albumin, SIGMA, A4503)). ), 1.08 μg / μl (Sample 1).

  The sample 1 is diluted with 100 mM SPB, and the protein concentrations are 1 ng / 10 μl (sample 1-1), 10 ng / 10 μl (sample 1-2), and 100 ng / 10 μl (sample 1-3), respectively. Samples were prepared.

  On the other hand, when the protein concentration of colostrum before enzyme treatment was determined in the same manner, it was 14.41 μg / μl (Comparative Sample 1). The comparative sample 1 was diluted with 100 mM SPB, and the protein concentrations were 1 ng / 10 μl (comparative sample 1-1), 10 ng / 10 μl (comparative sample 1-2), and 100 ng / 10 μl (comparative sample 1-3), respectively. ) Was prepared.

2. Macrophage phagocytic activity (1)
Mice (8 weeks old, ICR female, Japan SLC Co., Ltd.) were dislocated from the cervical vertebrae, peeled off the abdominal skin, and injured the internal organs. Phosphate buffered saline (PBS: 0.01M) Of sodium phosphate, 0.9% NaCl and 5 units / ml heparin) was injected. After tapping the abdomen for about 1 minute, peritoneal fluid was collected and peritoneal cells were collected. The collected peritoneal fluid was centrifuged (1500 rpm, 4 ° C., 15 minutes), the supernatant was discarded, RPMI medium was added, and pipetting was performed. The number of cells was counted with a Burker-Turk type hemocytometer, and further RPMI medium was added to adjust to 1.0 × 10 ⁶ cells / ml. The RPMI medium was prepared by the following operation. That is, in a clean bench, a powder medium (manufactured by GIBCO, catalog number: 856846) was dissolved in 900 ml of purified water, and then 2 g of NaHCO ₃ was further dissolved. The mixture was adjusted to pH 7.2 with 1N HCl and then made up to 1000 ml with purified water. The solution thus obtained was filtered through a filter (MILLIPORE, SLGVJ13SL) as RPMI medium and stored at 4 ° C. until use.

In a 24-well plate (TPP, 92024) containing three sterilized cover glasses (MATSANAMI, micro cover glass, No. 1) per well, the macrophage solution obtained above was added at 500 μl / well (5.0 ×). 10 ⁵ cells / well), and 500 μl / well of RPMI medium was further added to each well so that the total amount was 1 ml / well. After incubating the plate at 37 ° C. for 1 hour, the liquid in each well was discarded, and each well was washed twice with 1 ml of RPMI medium. After washing, an additional 1 ml of RPMI medium was added to each well and incubated at 37 ° C. for 15 hours.

  After incubation, 10 μl of each of the specimens 1-1 to 1-3 and the comparative specimens 1-1 to 1-3 prepared above was added to each well. Macrophages were stimulated by incubation at 37 ° C. for 3 hours. After incubation, the solution portion of each well was discarded, 1 ml of 0.5% opsonized SRBC (sheep erythrocyte, Japan Biomaterials Center) was added, incubated at 37 ° C. for 90 minutes, and phagocytosed by macrophages. After phagocytosis, the cover glass was washed sequentially with 1/5 × PBS, 1 × PBS, and 1 × PBS, and air-dried for about 30 minutes. After air drying, each cover glass was immersed in methanol (Kanto Chemical Co., Ltd., 25183-2B) for about 1 minute and fixed with methanol. After the fixation, it was air-dried again for about 30 minutes, and stained with Giemsa solution (SIGMA, A1327) diluted 20-fold with PBS for 1 hour. After dyeing, it was washed from the back side of the cover glass with tap water and air-dried overnight.

  After air drying, the cover glass was turned over and attached to a slide glass (MATSANAMI, micro slide glass, S2215). Take a 9-point photograph of each cover glass with an optical microscope (Nikon ECLIPSE E200), count the total number of macrophages observed, the number of phagocytosed SRBCs, the number of macrophages phagocytosed, and total the measurement values for 9 points. The ratio of macrophages that phagocytosed SRBC out of all the measured macrophages was multiplied by the average phagocytosis number of one macrophage to calculate the eating index. For reference, FIG. 1 shows a photograph after Giemsa staining showing the state of “phagocytic macrophages” and “phagocytic SRBC”. Macrophages are observed as purple spheres and SRBC as transparent spheres by Giemsa staining. The feeding index was calculated as SRBC phagocytosed SRBC in contact with macrophages and macrophages phagocytosed in macrophages in contact with SRBC.

  For each specimen, a feeding index of 2 to 3 was calculated for each cover glass, and the average value was obtained. As a control, the same operation as described above was performed using RPMI medium instead of the sample or the comparative sample. Two feeding indices were obtained for Comparative Samples 1-1 and 1-2, and three feeding indices were calculated for the rest.

  The results are shown in Table 1 (mouse peritoneal macrophage phagocytic activity using opsonized SRBC) and FIG.

3. Phagocytosis activity of intestinal macrophages (preparation of each sample)

  The LPS sample was prepared by diluting LPS (Lipopolysaccharide, from Escherichia coli) (manufactured by SIGMA, L2755) to 100 μg / ml with 100 mM PBS (pH = 7.0). The LPS sample activates intestinal macrophages as described in the literature (Seminars Immunopathology, 31 (2), 178-84, 2009).

  Serum samples were prepared by diluting untreated human serum to 100 μg / ml with 100 mM PBS (pH = 7.0).

  A human serum enzyme-treated sample is obtained by treating human serum with a human serum enzyme-treated product obtained by treating human serum in the same manner as the method for bovine colostrum according to the method of Patent Document 3 (International Publication No. 2013/038997). And diluted to 100 μg / ml with 100 mM PBS (pH = 7.0).

  Colostrum samples were prepared by diluting untreated bovine colostrum with 100 mM PBS (pH = 7.0) to 100 μg / ml.

  The bovine colostrum enzyme-treated sample was prepared by diluting bovine colostrum enzyme-treated product with 100 mM PBS (pH = 7.0) to 100 μg / ml.

(Preparation of medium)
In 17 ml of RPMI medium, 2 ml of collagenase D (Roche, 110888580001) and 1 ml of DNase I (Roche, 112883932001) were dissolved and warmed to 37 ° C. to prepare a collagenase medium.

(Measurement of phagocytic activity)
C57BL / 6 female mice (7 weeks of age) were anesthetized by intraperitoneal administration of 400 μl of 20 mg / ml chloral hydrate (sigma, A2374). The right abdomen of the mouse was cut, the intestine was exposed and each sample (1 mg / kg) was administered, and then the abdomen was closed. One hour after administration, the abdomen was cut again to expose the intestine, and unlabeled OVA protein (SIGMA, A5503-1G) and AF488-labeled OVA protein (life technologies, O34781) were administered, and the abdomen was closed. One hour after administration, the mice were dislocated from the cervical spine, and the intestines were removed. Fat and Peyer's patches were removed without damaging the removed intestine and washed with PBS. FACS buffer (phosphate buffered saline (PBS: 0.01 M sodium phosphate, 0.9% NaCl and 5 units / ml heparin) was cut to about 2 cm and warmed to 37 ° C. 41.98 ml, FBS (immobilized) (GIBCO, 10437) 5 ml, EDTA (Nacalai Tesque, 15111-45, 500 mM) 1 ml, HEPES (MP, 1688449, 1M) 1 ml, sodium pyruvate (GIMCO, 11360-070, 100 mM) 500 μl, polynexin sulfate B (GIMCO, 21850-029, 25 mg / ml) 20 μl, penicillin streptomycin (GIMCO, 15140-122 10,000 U / ml), 500 μl) in 50 ml, 37 ° C. C. in an incubator, a stirrer, and stirred for 20 minutes (about 250 rpm).

  After stirring, the intestine was taken out and washed 3 times with 30 ml of PBS. The intestines were transferred to a 10 cm diameter dish containing 10% FBS / RPMI medium. 4 ml of collagenase medium was added and the intestine was cut. 11 ml of collagenase medium was added and allowed to stand. The bubbles and fat that floated were removed with an aspirator. The tissue piece in the vial was transferred to a flask, the vial was washed with 5 ml of collagenase medium, and the washing solution was transferred to the flask. The flask was placed in a 37 ° C. incubator and the contents were stirred for 1 hour. After stirring, 400 μl of 0.5 M EDTA (pH = 8.0, prepared with PBS) was added to the flask, and the mixture was further stirred for 5 minutes. After stirring, the supernatant was transferred to a tube on which a cell strainer (FALCON, 352360) was placed. On the other hand, 10 ml of FACS buffer warmed to 37 ° C. was added to the tissue piece and suspended. The entire suspension was passed through a cell strainer and the remaining debris was squeezed out. The filtrate was centrifuged at 20 ° C. and 1800 rpm for 10 minutes. After centrifugation, the supernatant was removed and the cells were dispersed. The cells were suspended in 10 ml of 40% percoll, the suspension was transferred to a tube, 5 ml of 75% percoll was placed at the bottom using a capillary, and the tube was centrifuged at 2000 rpm at 20 ° C. for 20 minutes. After centrifugation, about 7 ml of 40% percoll was removed from the top of the tube with an aspirator. After removal, about 6 ml was added to a tube containing 5 ml of FACS buffer, and FACS buffer was added to a total volume of 14 ml, and the tube was centrifuged at 1800 rpm at 20 ° C. for 10 minutes. After centrifugation, the supernatant was removed, 2 ml of FACS buffer was added and suspended, and 1 ml was transferred to another tube. The tube was centrifuged at 20 ° C. and 1500 rpm for 3 minutes.

After centrifugation, the supernatant was removed, after addition of FACS buffer 2 ml, further Pacific (Pacific ^TM) anti-mouse F4 / 80 antibody (anti-mouse F4 / 80 Antibody ) (Biolegend, 123124), PE / cy7 antimouse / Human CD11b antibody (PE / cy7 ari-mouse / human CD11b Antibody) (Biolegend, 101216) and CD16 / 32 antibody (CD16 / 32 Anti-body) (BD, 553141) were added and reacted at 4 ° C. After 15 minutes, the supernatant was removed, 2 ml of a wash buffer was added, and the mixture was centrifuged at 20 ° C. and 1500 rpm for 3 minutes. After centrifugation, 200 μl of 1 μg / ml 7-aminoactinomycin D (sigma, A9400) solution was added, and the phagocytic activity of intestinal macrophages was measured by flow cytometry.

(result)
The results are shown in Table 2. The colostrum enzyme-treated product further increased the phagocytic activity of ovalbumin (OVA) -positive macrophages in the intestinal tract compared to the serum enzyme-treated product.

4). Molecular weight of active protein (HPA positive) in colostrum enzyme-treated product and serum enzyme-treated product (sample preparation)
As the marker 1, Novex (registered trademark) Sharp Pre-stained Protein Standard (Invitrogen ^™ , 745065) was used.

  As the serum, untreated human serum was diluted to 1 mg / ml with 100 mM PBS (pH = 7.0).

  As the serum enzyme-treated product, a human serum enzyme-treated product obtained in the same manner as above was diluted to 1 mg / ml with 100 mM PBS (pH = 7.0).

  As the marker 2, WIDE-VIEW Prestained Protein Size Marker III (manufactured by Wako, 230-02461) was used.

  As colostrum, untreated bovine colostrum was diluted to 1 mg / ml with 100 mM PBS (pH = 7.0).

  As the colostrum enzyme-treated product, bovine colostrum enzyme-treated product was diluted to 1 mg / ml with 100 mM PBS (pH = 7.0).

  Each sample was prepared to 1 μg / μl with distilled water, 5 μl of each sample, Sample buffer (950 μl of Laemmli Sample Buffer (manufactured by BIO-RAD, 161-0737), 50 μl of 2-mercaptoethanol (manufactured by SIGMA, A2029) was added) and 5 μl was mixed and heat-treated at 100 ° C. for 10 minutes to obtain a sample for electrophoresis.

  The electrophoresis gel (XV PANTERA GEL, DRC, NXV-381D20) is set in the electrophoresis tank (ERICA-MP, DRC, XVE-0MPC), and the electrophoresis buffer (high-speed SDS-PAGE electrophoresis buffer (DRV, NXV-BUPPTTG) is used. The electrophoresis tank was filled with a solution prepared by dissolving in 1000 ml of distilled water. Each sample for electrophoresis was applied to each well of the set gel, and electrophoresis was performed at 300V. The applied amount of each electrophoresis sample was 10 μl in lane 1, 1 μl in lanes 2 and 3, and 5 μl in lanes 4, 5, and 6. After completion of the electrophoresis, the upper part of the gel was cut off and set in a transfer device (MINICA-MP, DRC, XVE-0MPB). A PVDF membrane (BIO-RAD, 162-0177) immersed in methanol (Kanto Chemical Co., Ltd., 25183-2B) for 1 minute was placed on the gel and transferred at 47 V for 1 hour.

After transfer, the membrane was adjusted to pH 7.4 by dissolving TBS-T (8.0 g NaCl, 0.2 g KCL and 3.0 g H ₂ NC (CH ₂ OH) ₃ in 1000 ml distilled water. This was washed 3 times for 10 minutes with 1 ml of polyoxyethylene (20) sorbitan monolaurate (prepared by adding WAKO, 167-11515). The membrane was immersed in a blocking solution (prepared by dissolving 100 mg of BSA (bovine serum albumin, SIGMA, A4503) in 10 ml of TBS-T) and shaken at room temperature for 1 hour. The membrane was washed 3 times with TBS-T for 10 minutes, and 1 mg of primary antibody HPA (10 μl of HPA lectin (Lectin from Helix Pomatia biotin conjugate, lyophilized powder, L6512), 1 ml of 100 mM SPB) Was dissolved in 10 ml of TBS-T, and was shaken for 1 hour at room temperature. The membrane was washed 3 times with TBS-T for 10 minutes, immersed in the secondary antibody streptavidin (prepared by diluting 2 μl of streptavidin (GE healthcare, RPN-1231) with 10 ml of TBS-T). Shake for hours at room temperature. The membrane was washed 3 times with TBS-T for 10 minutes.

  In a plastic container, the membrane and ECL solution (1 ml of detection reagent 1 (ECL Western Blotting Detection Reagents, manufactured by GE healthcare, RPN2106V1) and 1 ml of detection reagent 2 (ECL Western Blotting Detection, manufactured by Regents, Inc. Prepared) was allowed to react for 1 minute and photographed with Lumicube (Liponics Corp., 5003). After imaging, the membrane was washed 3 times with TBS-T for 10 minutes, and immersed in CBB staining solution (PAGE Blue 83, Cosmo Bio Inc., 423406) for 10 minutes, and a protein band was observed (FIG. 3). From the results, the molecular weight of the active protein (HPA positive) in the serum enzyme-treated product was identified as 70, 55, 51, and 17 kDa, whereas the molecular weight of the active protein (HPA positive) in the colostrum enzyme-treated product was 245, It was identified as 82 kDa.

5. Preparation of bovine colostrum enzyme-treated product (2)
Except that sialidase was not used, the protein concentrations were 10 ng / 10 μl (sample 2-1) and 100 ng / 10 μl (sample 2- Each specimen of 2) was prepared. As a positive control, a sample with LPS of 1 μg / 10 μl was prepared.

6). Macrophage phagocytic activity (2)
Using the specimen, the feeding index was determined in the same manner as macrophage phagocytic activity (1). The results are shown in Table 3 (mouse peritoneal macrophage phagocytic activity using opsonized SRBC).

  According to the present invention, it is possible to provide a bovine colostrum enzyme-treated product and a method for producing the same, which are useful for treatment, prevention, improvement, maintenance of remission of diseases such as cancer and infectious diseases.

1 phagocytic macrophages 2 phagocytic SRBC
3 Macrophages

Claims (7)

For a single dose comprising contacting bovine colostrum with β-galactosidase in the presence of an amount of buffer necessary to adjust the total protein concentration in bovine colostrum, A method for producing a bovine colostrum enzyme-treated product exhibiting a macrophage activating action comprising a protein in the range of 40 mg / kg.   The production method according to claim 1, further comprising a step of contacting bovine colostrum with sialidase. Contacting bovine colostrum with β-galactosidase in the presence of an amount of buffer necessary to adjust the total protein concentration in bovine colostrum, for a single dose, from 0.02 μg to A method for producing a pharmaceutical composition comprising a bovine colostrum enzyme-treated product exhibiting a macrophage activating action comprising a protein in the range of 40 mg / kg.   The method for producing a pharmaceutical composition according to claim 3, further comprising the step of contacting bovine colostrum with sialidase.   The method for producing a pharmaceutical composition according to claim 3 or 4, which is used for anti-cancer or anti-infection. Contacting bovine colostrum with β-galactosidase in the presence of an amount of buffer necessary to adjust the total protein concentration in bovine colostrum, for a single dose, from 0.02 μg to A method for producing a composition for food and drink, comprising a bovine colostrum enzyme-treated product that exhibits a macrophage activating action containing a protein in the range of 40 mg / kg.   The manufacturing method of the composition for food-drinks of Claim 6 which further comprises the process which makes bovine colostrum contact with sialidase.

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